Cellular actin protrusions (e.g. filopodia, microvilli, and stereocilia) display a broad range of lengths and lifetimes critically related to their specific cellular function. Stereocilia, the mechanosensory organelles of hair cells, are a distinctive class of actin-based cellular protrusions with an unparalleled ability to regulate their lengths over time. Our laboratory has made advances towards elucidating the mechanisms that underlie the formation, regulation, renewal, and life span of stereocilia. In earlier studies we have shown that in developing neonatal hair cells stereocilia actin filaments are continuously polymerizing and depolymerizing in a treadmilling mechanism that results in a continuous dynamic renewal while maintaining steady-state lengths. Whether stereocilia actin filaments are dynamically turning over throughout the lifetime of the hair cell, and whether mature stereocilia have any structural plasticity has remained an important open question in the hearing research field, especially since structural plasticity in hair cell stereocilia may have implications for the development of therapeutic interventions for both inherited and acquired hearing losses. In a recent study, we showed that two paralogs of class 3 myosins Myo3a and Myo3b transport the actin-regulatory protein Espin 1 (Esp1) to stereocilia and filopodia tips in a remarkably similar, albeit non-identical fashion. We followed up this study with experimental and computational data (in collaboration with Nir Gov, Weizmann Institute) that suggests that subtle differences between the biophysical and biochemical properties of these myosins can help us understand how they target and regulate the lengths of actin protrusions. Through the combination of experimental and computational approaches, we were able to dissect the underlying molecular components of these myosin:cargo dynamics. The work demonstrated the intricate nature of the interactions and the usefulness of theoretical modeling in deciphering them. Indeed, these results strongly indicate that Myo3b can behave quite similarly to Myo3a, but they also show that Myo3b falls short of Myo3a in terms of elongation activity. This finding is one plausible explanation for the late-onset phenotype associated with mutations in MYO3A. Overall, these studies lend stronger support for further exploring the potential role for Myo3b in compensating for Myo3a, and how these two motor proteins interact in a physiological context. We have also been using an in vivo transgenic mouse approach to elucidate the role of Myo3a, Myo3b, and Esp1. We observed that mice lacking Myo 3a or Myo3b individually show no stereocilia shape changes. While mice lacking Esp1 show a subtle stereocilia length differences instead of the characteristic staircase pattern. This lead us to the hypothesis that there are other yet to be identified cargo for Myo 3a and Myo3b that compensate for Esp1. A immuncytochemical screen for other stereocilia proteins showed that a protein very similar to Esp1 is indeed localized at stereocilia tips in a pattern analogous to Esp1. We are now pursuing the characterization of this protein and its role in stereocilia length regulation. In another study in collaboration with Nir Gov we tested a reaction diffusion concept to describe the initiation of actin-based protrusions, and compared with experimental observations. Reaction-diffusion models have been used to describe pattern formation on the cellular scale, and traditionally do not include feedback between cellular shape changes and biochemical reactions. In this study we introduce a distinct reaction-diffusion-elasticity approach: The reaction-diffusion part describes bi-stability between two actin orientations, coupled to the elastic energy of the cell membrane deformations. This coupling supports spatially localized patterns, even when such solutions do not exist in the uncoupled self-inhibited reaction-diffusion system. This concept was applied to describe the nonlinear (threshold driven) initiation mechanism of actin-based cellular protrusions and provide support by several experimental observations. In a another study, in collaboration with Dr. Walter Marcotti from Sheffield University, we showed that the actin-binding protein epidermal growth factor receptor pathway substrate 8 (Eps8)L2, a member of the Eps8-like protein family, is a newly identified hair bundle protein that is localized at the tips of stereocilia of both cochlear and vestibular hair cells. It has a spatiotemporal expression pattern that complements that of Eps8. In the cochlea, whereas Eps8 is essential for the initial elongation of stereocilia, Eps8L2 is required for their maintenance in adult hair cells. In the absence of both proteins, the ordered staircase structure of the hair bundle in the cochlea decays. In contrast to the early profound hearing loss associated with an absence of Eps8, Eps8L2 null-mutant mice exhibit a late-onset, progressive hearing loss that is directly linked to a gradual deterioration in hair bundle morphology. We conclude that Eps8L2 is required for the long-term maintenance of the staircase structure and mechanosensory function of auditory hair bundles. It complements the developmental role of Eps8 and is a candidate gene for progressive age-related hearing loss. Age-related hearing loss and noise-induced hearing loss are major causes of human morbidity. In collaboration with Dr. Xue Z. Liu, University of Miami, we showed that a shared cause of these disorders may be loss of function of the ATP-gated P2X2 receptor (ligand-gated ion channel, purinergic receptor 2) that is expressed in sensory and supporting cells of the cochlea. Genomic analysis of dominantly inherited, progressive sensorineural hearing loss DFNA41 revealed a p.V60L mutation, which co-segregated with fully penetrant hearing loss. We helped demonstrate that this mutation abolishes two hallmark features of P2X2 receptors: ATP-evoked inward current response and ATP-stimulated permeability, measured as loss of ATP-activated FM1-43 fluorescence labeling. Co-expression of mutant and WT P2X2 receptor subunits significantly reduced ATP-activated membrane permeability. P2X2-null mice developed severe progressive hearing loss, and their early exposure to continuous moderate noise led to high-frequency hearing loss as young adults. Similarly, among family members heterozygous for P2X2 p.V60L, noise exposure exacerbated high-frequency hearing loss in young adulthood. Our collective results suggest that P2X2 function is required for life-long normal hearing and for protection from exposure to noise. Sensory processing in the auditory system requires that synapses, neurons, and circuits encode information with particularly high temporal and spectral precision. In collaboration with Dr. Henrique von-Gersdorf, Vollum Institute, we combined serial electron microscopy, paired physiological recordings and numerical simulations to examine novel mechanisms that facilitate fast frequency-tuned synaptic transmission in the inner ear. Three-dimensional anatomical reconstructions reveal specialized spine-like contacts between individual afferent fibers and hair cells that are surrounded by large, open regions of extracellular space. Morphologically realistic diffusion simulations suggest that these local enlargements in extracellular space speed transmitter clearance and reduce spillover between neighboring synapses, thereby minimizing postsynaptic receptor desensitization and improving sensitivity during fast frequency-tuned signaling. Accordingly, evoked excitatory postsynaptic currents in afferent fibers are unaffected by glutamate transporter blockade, indicating that transmitter diffusion and dilution, not uptake, ensures fidelity at these synapses.